Among the plasmids found in Pseudomonas aeruginosa that contribute to its antibiotic resistance and consequent success as a nosocomial pathogen, those belonging to the P-2 imcompatibility group are of particular interest because of their prevalence, ability to mediate resistance to the otherwise effective anti-Pseudomonas drugs amikacin, carbenicillin, gentamicin, and tobramycin, and their recently discovered huge size of 280 to 312 megadaltons. This project is for studies on the physical and genetic organization of P-2 plasmids, which also include the metabolic plasmids CAM and OCT found in soil pseudomonads. P-2 plasmid DNA will be purified after cell lysis and alkali denaturation by ethidium bromide-cesium chloride gradient centrifugation. Purified plasmid DNA will be analyzed by restriction endonuclease cleavage and agarose gel electrophoresis. A derivative of the multicopy, broad host range plasmid R1162 will be used as a vector for clonong P-2 DNA with the aim of developing a complete restriction map of the parent plasmid from cloned fragments in piece-wise fashion. Detailed localization will be accomplished by transposon-induced insertional inactivation and deletion formation. The structure of different IncP-2 R plasmids and of P-2 R and metabolic plasmids will be compared to determine relationships within this interesting plasmid group.